Culture of human bone marrow-derived mesenchymal stem cells on of poly(L-lactic acid) scaffolds: potential application for the tissue engineering of cartilage.

PURPOSE: Due to the attractive properties of poly(L-lactic acid) (PLLA) for tissue engineering, the aim was to determine the growth and differentiation capacity of mesenchymal stromal cells (MSCs) in PLLA scaffolds and their potential use in the treatment of cartilage diseases. METHODS: MSCs were cultured in PLLA films and thin porous membranes to study adherence and proliferation. Permeability and porosity were determined for the different scaffolds employed. The optimal conditions for cell seeding were first determined, as well as cell density and distribution inside the PLLA. Scaffolds were then maintained in expansion or chondrogenic differentiation media for 21 days. Apoptosis, proliferation and chondrogenic differentiation was assessed after 21 days in culture by immunohistochemistry. Mechanical characteristics of scaffolds were determined before and after cell seeding. RESULTS: MSCs uniformly adhered to PLLA films as well as to porous membranes. Proliferation was detected only in monolayers of pure PLLA, but was no longer detected after 10 days. Mechanical characterization of PLLA scaffolds showed differences in the apparent compression elastic modulus for the two sizes used. After determining high efficiencies of seeding, the production of extracellular matrix (ECM) was determined and contained aggrecan and collagens type I and X. ECM produced by the cells induced a twofold increase in the apparent elastic modulus of the composite. CONCLUSIONS: Biocompatible PLLA scaffolds have been developed that can be efficiently loaded with MSCs. The scaffold supports chondrogenic differentiation and ECM deposition that improves the mechanics of the scaffold. Although this improvement does not met the expectations of a hyaline-like cartilage ECM, in part due to the lack of a mechanical stimulation, their potential use in the treatment of cartilage pathologies encourages to improve the mechanical component.

Structure, regulation, and physiological functions of NADPH oxidase 5 (NOX5).

NOX5 is the last member of the NADPH oxidase (NOXs) family to be identified and presents some specific characteristics differing from the rest of the NOXs. It contains four Ca2+ binding domains at the N-terminus and its activity is regulated by the intracellular concentration of Ca2+. NOX5 generates superoxide (O2•-) using NADPH as a substrate, and it modulates functions related to processes in which reactive oxygen species (ROS) are involved. Those functions appear to be detrimental or beneficial depending on the level of ROS produced. For example, the increase in NOX5 activity is related to the development of various oxidative stress-related pathologies such as cancer, cardiovascular, and renal diseases. In this context, pancreatic expression of NOX5 can negatively alter insulin action in high-fat diet-fed transgenic mice. This is consistent with the idea that the expression of NOX5 tends to increase in response to a stimulus or a stressful situation, generally causing a worsening of the pathology. On the other hand, it has also been suggested that it might have a positive role in preparing the body for metabolic stress, for example, by inducing a protective adipose tissue adaptation to the excess of nutrients supplied by a high-fat diet. In this line, its endothelial overexpression can delay lipid accumulation and insulin resistance development in obese transgenic mice by inducing the secretion of IL-6 followed by the expression of thermogenic and lipolytic genes. However, as NOX5 gene is not present in rodents and human NOX5 protein has not been crystallized, its function is still poorly characterized and further extensive research is required.

NADPH Oxidase 5 Induces Changes in the Unfolded Protein Response in Human Aortic Endothelial Cells and in Endothelial-Specific Knock-in Mice.

Oxidative stress constitutes a key molecular mechanism in the development of cardiovascular diseases. A potential relationship between reactive oxygen species (ROS) driven by the NADPH oxidase family (NOX) and the unfolded protein response (UPR) has been postulated. Nevertheless, there is a lack of information about the crosstalk between NOX5 homologue and the UPR in a cardiovascular context. The main aim was to analyze NOX5-mediated ROS effects in the UPR and its importance in cardiovascular diseases. To this effect, we used an adenoviral NOX5-ß overexpression model in human aortic endothelial cells (HAEC) and a conditional endothelial NOX5 knock-in mouse. Using expression arrays, we investigated NOX5-induced genomic changes in HAEC. Compared with the control HAEC, 298 genes were differentially expressed. Gene ontology analysis revealed the activation of numerous cellular routes, the most relevant being the UPR pathway. Using real-time PCR and Western Blot experiments, we confirmed that NOX5 overexpression induced changes in the expression of the UPR components, which were associated with increased apoptosis. Moreover, in endothelial-specific NOX5 knock-in mice, we found changes in the expression of the UPR components genes. In these mice, myocardial infarction was performed by permanent coronary artery ligation; however, NOX5 expression was not associated with differences in the UPR components mRNA levels. In these animals, we found significant associations between the UPR components gene expression and echocardiographic parameters. Our data support the idea that NOX5-derived ROS may modulate the UPR pathway in endothelial cells, which might play a relevant role in cardiac physiology.

Gene expression and proliferation analysis in young, aged, and osteoarthritic sheep chondrocytes effect of growth factor treatment.

Cartilage is a support tissue with a poor capacity to self-repair. Its cells, chondrocytes, are responsible for synthesizing and renewing the matrix that surrounds them in a constant turnover mechanism. Autologous chondrocyte implantation (ACI) is one of the techniques that promises to be an alternative to common strategies for chondral lesions. To apply this technique, a large amount of cells must be obtained. In our work, we studied the state of cells from different cartilage (young, aged, and osteoarthritic sheep) cultured in monolayer by analyzing their proliferation rate using bromodeoxyuridine and their gene expression profile by RT-PCR. A decrease was found in expression of type II collagen and aggrecan in aged, osteoarthritic, and passaged chondrocytes. Treatment of cells with growth factors aFGF, IGF-I, TGF-beta, and OP-1 improved the proliferation rate in all cells studied and stimulated gene expression of type II collagen, aggrecan, and TGF-beta. Osteoarthritic cells showed a poor response according to matrix gene expression, while young cells responded properly, and aged chondrocytes showed a moderate response. These results suggest that the state of cartilage may affect the behavior of cultured chondrocytes.

Isolation, culture and characterization of adult carotid body-derived cells.

Recent studies indicate that carotid body (CB) could be a suitable cell source for cell therapy in Parkinson's disease. We have isolated and successfully expanded in culture as monolayer adult CB-derived cells using a modification of the culture medium employed for bone marrow multipotent adult progenitor cells (MAPCs). These cells express variable amounts of tyrosine hydroxylase (TH), beta-III tubulin and Sox2. In addition, CB-derived cells showed high expression of Sox2 related to a high rate of proliferation and consistent with an undifferentiated state. Under culture conditions that reduced cell proliferation, Sox2 expression decreased while TH and beta-III tubulin expression was increased. This could indicate that the differentiation of some cells occurs in the culture, thus accounting for a certain neural differentiation potential of CB-derived cells.

In vitro healing of avascular meniscal injuries with fresh and frozen plugs treated with TGF-beta1 and IGF-1 in sheep.

We studied the effect of freezing and inserting meniscal plugs in lesions generated in the avascular area of sheep menisci maintained in vitro, and whether the healing process can be improved by adding growth factors TGF-beta1 and IGF-1. Thirty six menisci obtained from healthy 3 months-old sheep were cultured in 6 well plates and holes were perforated in the avascular area. Meniscal plugs, either fresh or frozen at -20 degrees C for 1 month, were used to fill in the lesions, and then cultured in the presence or absence of TGF-beta1 or IGF-1 for 8 weeks. Samples stained with Massons trichrome were analyzed to evaluate the attachment of the plug and the cell density of the tissue. BrdU immunohistochemistry was performed to identify the proliferation of meniscal cells. Both growth factors improved considerably the cell density of implanted plugs. TGF-beta1 increased significantly the attachment of both fresh and frozen plugs, but it had no effect on meniscal cell proliferation. In contrast, IGF-1 had no effect on the attachment, but did increase significantly the number of proliferating cells in the surface of the host meniscus and the inserted plug. In conclusion, frozen plugs can survive if treated with either TGF-beta1 or IGF-1. The combination of TGF-beta1 and IGF-1 could aid in the repairing of the avascular meniscal injuries, as they are capable of promoting the attachment of tissue, and increasing the proliferation of meniscal cells.

IGF-1 gene therapy to protect articular cartilage in a rat model of joint damage.

INTRODUCTION: An adeno-associated virus (AAV) derived vector in gene transfer model that induces IGF-1 expression could repair articular cartilage. MATERIALS AND METHODS: Male Wistar rats, 150 and 200 g, and 7 weeks old, were used. Effectiveness of constructed vectors was assayed inoculating them in rat knees of control and damaged animals either mechanically or by collagen-induced arthritis. Inoculation was intra-articular with 50 microL of recombinant AAV-Luciferase (1.25 x 10(8) particles). The rats were killed after 1, 2, 4 and 8 weeks. IGF-I activity was analyzed by injecting 50 microL of recombinant AAV (1.25 x 10(8) particles) in animals with damaged knees. Final analysis was performed after 8 weeks. RESULTS: The activity of AAV vectors in vitro shows the presence of mRNA coding to IGF-I in cells infected with AAV-IGF and not in control cells without viral vectors and an increase in secreted IGF-I protein in culture medium. In vivo, AAV derived vectors induced protein expression in cartilage 2 months after inoculation. In the animals killed after 1 and 2 weeks, no significant increase in the reaction of luciferase was observed (P > 0.05). In the group of animals with no injury an increase was observed at 4 weeks, which was more marked and significant after 8 weeks (P = 0.029). The same behavior occurred in the animals with induced arthritis and in the mechanical injury group. In the levels of expression after 8 weeks, no significant differences were found between the two groups of injured animals and the group of healthy animals infected with the virus. The joints of the animals that were subjected to injuries in the cartilage and inoculated with AAV-IGF-I presented a similar appearance to those animals inoculated with saline solution. CONCLUSION: Autoimmune and mechanical lesions did not show improvement in the state of its cartilage after the treatment. The use of AAV vectors capable of inducing the expression of IGF-I in vitro is therefore not sufficient to protect the cartilage from the serious damage.

Cell viability and protein composition in cryopreserved cartilage.

Chondrocyte survival in frozen-stored osteochondral allografts is low. We analyzed different storage conditions to determine the best for maintenance or storage for preserving cartilage. We hypothesized cell viability and extracellular protein content could be improved with better cryopreservation. Articular cartilage from nine sheep femoral condyles were stored at 4 degrees C, -80 degrees C, and -196 degrees C with and without cryopreservative agents for 1 month. We determined cell viability and performed Western blot analysis for TGF-beta, IGF-1, and MMP-2, 9, and 13. We observed decreases in viability in cartilage stored at 4 degrees C: 36.2% viability when stored without solution, 40.4% in phosphate-buffered saline, and 48.1% in culture medium. TGF-beta and IGF-1 decreased in the first week in all groups. The groups stored at -80 degrees C and -196 degrees C contained less protease in the last 2 weeks. In the groups stored at 4 degrees C in phosphate-buffered saline, we detected no increase in the levels of MMPs. Our data discourage the use of frozen cartilage because of the decrease of cell viability and elevation in MMPs. However, modifying the freezing conditions might moderate these changes and improve the state of preserved cartilage.

Suppression of angiogenesis and tumor growth by adenoviral-mediated gene transfer of pigment epithelium-derived factor.

Pigment epithelium-derived factor (PEDF) was identified from retinal pigment epithelial cells and has been shown to display neurotrophic effects. In addition it has been found to induce a potent inhibition of angiogenesis. In this study we have explored whether overexpression of PEDF by a gene transfer approach can block tumor angiogenesis and reduce tumor growth. We found that cells infected with an adenovirus encoding PEDF under the control of the CMV promoter (AdPEDF) secreted PEDF protein into the medium that exhibited strong inhibitory effects on migration and tube formation of endothelial cells cultured in the presence of vascular endothelial growth factor. Moreover, the systemic administration of AdPEDF was able to inhibit angiogenesis in Matrigel assay in vivo, and treatment with this adenovirus of established hepatocellular carcinoma tumor in nude mice resulted in strong suppression of tumor growth. This anti-tumor effect could also be seen in a mouse lung carcinoma model by systemic administration of vector. In that model, treatment of tumor by intratumoral injection of AdPEDF also caused significant inhibition of tumor growth. The anti-tumor effect was related to a decrease in density of microvessels in tumors after treatment with AdPEDF. These data suggest that the antiangiogenic properties of PEDF can be exploited to inhibit the establishment of tumor neovasculature and reduce tumor growth.